Abstract

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Our laboratory has recently discovered that human 06-methylguanine DNA methyltransferase (MGMT), a unique DNA repair protein and an anticancer target, interacts with replication components (e.g. PCNA), and is also an integral part of the replication licensing system (ORCs/Cdt1/geminin etc.) that ensures no part of the genome is replicated more than once in the S phase. The free living nematode, Caenorhabditis elegans offers an elegant system to study MGMT's role in licensing control, and how it interfaces with differentiation and developmental programs at an organism level. C. elegans has two distinct orthologues of MGMT, known as AGT-1 and AGT-2, about which little is known. To this end, we first established an RNAi protocol to deplete either gene and compared the F1 progeny against wild type for differences in DNA synthesis and embryo development. Second, using O6-benzylguanine, a specific inhibitor of the AGT proteins, we established an embryo counting assay to determine brood size of the worms. Finally, using microscopic techniques (phase contrast and immunofluorescence), we have shown that AGT depletion causes gross morphological changes in the worm. Our preliminary data suggests that both AGT genes play a role in embryogenesis and germ line differentiation, and that loss of either gene results in fewer embryos in the fertile adult worms. Our other studies have shown that MGMT inactivation inhibits DNA synthesis and MGMT overexpression in human tumor cells induces polyploidy. Ongoing experiments to elucidate MGMT's role in replication licensing of chromosomal DNA will be described (Supported by RO1 CA 97343 grant to KSS and the Mirick-Myers Endowment in Geriatric Medicine of TTUHSC, Amarillo ).